Periodontal diseases, such as gingivitis and periodontitis, involve chronic inflammation in the gingival tissue caused by microbial communities and host immune responses. They are one of the most ubiquitous diseases worldwide, and remain the most common cause of tooth loss in the world today, and can affect up to 90% of the population worldwide. Gingivitis is defined per the FDA monograph (12 CFR Part 356, Vol. 68, No. 103 (2003)) as “An inflammatory lesion of the gingiva that is most frequently caused by dental plaque. Gingivitis is characterized by tissue swelling and redness, loss of stippling (a normal state in which the surface of healthy gingiva is comprised of small lobes), glossy surface, and increased tissue temperature. The gingiva also may bleed upon gentle provocation, such as tooth brushing or may bleed spontaneously. Gingivitis is usually not painful.” In healthy gingiva, the microbial community is in a homeostatic equilibrium with the host, and host immune systems limit bacterial overgrowth and neutralize toxic products, such as lipopolysaccharides (LPS) and lipoteichoic acids (LTA). The intricate balance between host and bacteria is disrupted as bacteria overgrow in the gingival margins or in the subgingival crevice. Recent data from metagenomics studies showed that bacterial species were increased in supragingival and subgingival plaques, such as Prevotella pallens, Prevotella intermedia, Porphyromonas gingivalis, and Filifactor alocis. Although the etiology of gingivitis and periodontitis remains elusive, one thing is clear; the composition of the dental plaques is significantly different in healthy sites compared with clinically defined disease sites. This observation, together with advances in characterizing the host and bacterial interactions using the newly developed tools in genomics, proteomics and metabonomics, has led to the notion that gingivitis and periodontitis are the result of disrupted homeostasis between host and polymicrobial communities (Lamont R J and Hajishengallis G. Polymicrobial synergy and dysbiosis in inflammatory disease. G Trends Mol Med. 2015; 21:172-83).
Polymicrobial communities in the dental plaques produce various virulence factors; for example, many bacteria produce digestive enzymes, such as hyaluronidases to breakdown polysaccharides that glue the host cell together, fibrinolytic enzymes that lyse the fibrins of blood clots, and collagenases that degrade collagens in the connective tissues. Gram negative bacteria secrete endotoxins, also called LPS, while Gram positive bacteria produce LTA and peptiglycans. Furthermore, one pathogen bacterium can generate multiple virulence factors; for example P. gingivalis has been reported to generate multiple virulence factors that are involved in the inflammatory and destructive events of periodontal tissues. These influence factors include the capsule, outer membrane, its associated LPS, fimbriae, proteinases, and selected enzymes.
LPS is an integral component of all Gram negative bacteria and is found in the outer membrane layer. P. gingivalis LPS possesses significant amounts of heterogeneity containing tetra- and penta-acylated structures. Several of them have been purified. LPS 1690 is highly toxic, while LPS 1435/1449 is relatively mild. Chemically, LPS consists of a hydrophilic polysaccharide and a hydrophobic lipid moiety referred to as lipid A. The latter is the actual toxic moiety of the LPS molecule and contains phosphate groups shown to be essential for its proinflammatory activity. Mechanistically, LPS first binds to LPS-binding protein (LBP), then the LBP-LPS complex is transferred to membrane-bound CD14, thereby enabling interactions with Toll-like receptor (TLR) 4 on cell membranes. Binding of LPS to TLR4 on the cell membrane activates both TIRAP-MyD88-dependent NFkB and TRAM-TRIF-dependent IRF3 or IRF7 signaling pathways, and subsequently stimulate production of proinflammatory cytokines and chemokines, such as interferon (IFN) gamma, tumor necrosis factor-α (TNFα), interleukin (IL)-1β, IL-6, IL-8, and IL-12. Also, induced is production of nitric oxide, prostaglandins, leukotrienes, and proteolytic enzymes. Importantly, LPS has been reported to cause periodontitis in mouse and rats.
P. gingivalis also secretes exotoxins and enzymes that exert damage on the host following their release. These enzymes include proteases, coagulases, and fibrinolysins. Noticeably, P. gingivalis generates peptidylarginine deiminase that can modify free or peptide-bound arginine to citrulline. The citrullinated proteins are especially harmful since they cause auto-immune responses, and are hypothesized to be the culprit of rheumatoid arthritis. In addition, P. gingivalis also produces two types of gingipains, lysine specific (Kgp) and arginine specific (Rgps). Gingipains play a major role in stirring up inflammation and tissue destruction in the periodontium.
Peptidoglycan is the cell wall component common to all Gram-negative and Gram-positive bacteria. It is a polymer consisting of sugars and amino acids that form a mesh-like layer outside the plasma membrane. The sugar component consists of alternating residues of β-(1, 4) linked N-acetylglucosamine and N-acetylmuramic acid. A peptide chain of three to five amino acids is cross-linked to the N-acetylmuramic acid. The peptide chain can also be cross-linked to the peptide chain of another strand of peptidoglycans to weave into a 3D mesh-like layer. The peptidoglycan layer is substantially thicker in Gram-positive bacteria (20 to 80 nanometers) than in Gram-negative bacteria (7 to 8 nanometers). Peptidoglycan accounts for around 90% of the dry weight of Gram-positive bacteria but only about 10% of Gram-negative strains. Thus, presence of high levels of peptidoglycan is the primary determinant of the characterization of bacteria as Gram-positive.
Both peptidoglycans and LTA have been shown to act as inflammatory mediators by activating TLR2 on the cell membrane of host innate immune cells and intracellular signaling receptors, such as nucleotide-binding oligomerization domain or NOD 1 and NOD 2. Binding to TLR2 activates the NF-κB signaling pathway, subsequently leading to production and release of proinflammatory cytokines and chemokines, such as IL-1α, IL-1β, IL-6, IL-8, IFN y, and TNF-α. Both Gram-negative and positive bacteria and the virulence factors (LPS, peptidoglycans and LTA) induce production of the inducible isoform of nitric oxide synthases. The latter catalyze the production of nitric oxide (NO) from L-arginine. NO is an important cellular signaling molecule, that promotes vascular dilation and many cellular functions. NO is also a free radical with an unpaired electron and is reported to kill bacteria. The inducible isoform of nitric oxide synthases is induced by LPS and other bacterial toxins, and is a part of innate immune responses.
As L-arginine is converted into NO by nitric oxide synthases, a byproduct, citrulline is regenerated. Citrulline is an amino acid that is not encoded in the genetic codes, so that is not incorporated in proteins during translation processes. Its name is derived from citrullus, the Latin word for watermelon. Citrulline is also a key intermediate in the urea cycle, the pathway by which mammals excrete ammonia. Citrulline is synthesized from ornithine and carbamoyl phosphate in the urea cycle, in which urea is produced in a series of reactions. Some of the reactions are carried out in the mitochondrial matrix and others in the cytosol.
The main metabolites of the urea cycle reactions are free amino acids, such as arginine, ornithine, citrulline, and arginisosuccinate. Arginine is the key intermediate in the urea cycle, and in NO production. It is cleaved by the cytosolic enzyme arginase, generating urea and ornithine. Ornithine, formed in the cytosol, is transported to the mitochondrial matrix via the action of ornithine translocase. In the mitochondria, ornithine transcabamoylase (OTC) catalyzes the condensation of ornithine with carbamoyl phosphate, producing citrulline. Concomitant with ornithine transport into the mitochondria is the export of citrulline to the cytosol where the remaining reactions of the cycle take place. Subsequently, citrulline is condensed with aspartate to form arginosuccinate, catalyzed by cytosolic argininosuccinate synthetase. Arginine and fumarate are produced from argininosuccinate by the cytosolic enzyme argininosuccinate lyase (also called argininosuccinase). The fumarate is reconverted to aspartate for use in the argininosuccinate synthetase reaction. In the final step of the urea cycle, arginases break arginine into urea and ornithine. The regenerated cytosolic ornithine is transported to the mitochondrial matrix for another round of urea synthesis. There are two arginase genes in humans, identified as the ARG1 and ARG2 genes. The ARG1 encoded isoform of arginase is a cytosolic enzyme primarily expressed in the liver and functions as the urea cycle enzyme. The ARG2 encoded arginase (arginase-2) is a mitochondrially localized enzyme expressed in non-hepatic tissues, primarily the kidney. The arginase-2 isoform is thought to be involved in nitric oxide and polyamine metabolism, however, the precise role of this enzyme is not clearly defined. More broadly, the biological functions of ornithine, citrulline, arginisosuccinate and arginine are not well understood yet in periodontal health.
Assessing the severity of gingivitis and periodontitis in a person is currently achieved with clinical measures such as gum redness, gum bleeding or pocket depth. While the measures are based on professionally developed scales, the actual values can vary due to examiner differences. There exists a need to quantify how severe gingivitis is and how effective treatments from oral hygiene products are in reducing the inflammatory response. It is desirable to have objective readings from an instrument that is free of human errors. Transcriptomics, proteomics and metabonomics measurements in saliva have been used to diagnose gingivitis, and to monitor progresses in treatment. But there is a disadvantage associated with saliva, in that the composition of saliva will be varied dependent upon the time of collection. As should be apparent, this field has a need for a more sensitive, accurate and consistent test whenever the volunteers appear in a dentist office, or in a clinical setting, or at home.